Lactid acid bacteria as priobiotic strains and compositions containing same

ABSTRACT

Useful probiotics have been selected among lactic acid bacteria strains of the genus  L. acidophilus, L. crispatus, L. gasseri, L. helveticus  and  L. jensenii  for their ability to kill urogenital and/or gastrointestinal pathogens and their ability to inhibit internalization of urogenital and/or gastrointestinal pathogens within urogenital and/or gastrointestinal epithelial cells in humans. Probiotic compositions comprise at least one of the said lactic acid bacteria strains in combination with a suitable delivery system, such as a food product or a beverage, a food or beverage compositions, a food or beverage supplement or adjuvant.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a U.S. National Phase of International ApplicationNo. PCT/EP2005/011152, filed Oct. 17, 2005, designating the U.S. andpublished in English on May 4, 2006 as WO 2006/045475, which claims thebenefit of International Application No.: PCT/EP2004/0119810, filed Oct.22, 2004; International Application No. PCT/EP2004/011981, filed Oct.22, 2004; and International Application No. PCT/EP2005/001354, filedFeb. 10, 2005.

FIELD OF THE INVENTION

This invention refers to the use of new probiotic bacteria stains in thetreatment of infectious troubles caused by various pathogens in mammals,more specifically the prevention and/or the treatment of urogenitaland/or gastrointestinal infections in humans.

BACKGROUND OF THE INVENTION

Urogenital infections remain a common problem, particularly in thefemale population. Bacterial adherence to the urogenital epithelium isrecognized as an important mechanism in the initiation and pathogenesisof urinary tract infections (UTI) and, in particular, of vaginalinfections. The urogenital pathogens originate predominantly in theintestinal tract and initially colonize the per-urethral region andascend into the bladder, resulting in symptomatic or asymptomaticbacterial uria. Alternatively, these bacteria invade and then colonizethe vagina causing there various types of symptomatic as well asasymptomatic vaginal infections. Thereafter, depending on host factorsand bacterial virulence factors, the organisms may further ascend andgive rise to pyelonephritis, respectively ascending infections of thegenital tract in women. Urogenital pathogens express virulencecharacteristics that enable them to resist the normally efficient hostdefence mechanisms.

The use of bacteria of the autochthonous flora, such as lactobacilli, toexclude urogenital pathogens from colonizing the urogenital tract is anestablished concept studied rather extensively since years (see e.g.Cadieux et al.—Lactobacillus strains and vaginal ecology—Jama.287:1940-41/2002; Butler B C, Beakley J W. Bacterial flora in vaginitis.Am J Obstet Gynaecol 1960; 78:432-40, Eschenbach D A, Davick P R,Williams B L, Klebanoff S J, Young-Smith K, Critchlow C M et al.Prevalence of hydrogen peroxide-producing Lactobacillus species innormal women and women with bacterial vaginosis. J Clin Microbiol 1989;27:251-6, Sobel J D, Cook R L, Redondo-Lopez V. Lactobacilli: a role incontrolling the vaginal microflora? in Horowitz B J, Mardh P-A, eds.Vaginitis and Vaginosis, pp 47-53. New York: Wiley-Liss, 1991, LauritzenC, Graf F, Mucha M. Restoration of the physiological vaginal environmentwith Doederlein bacteria and estriol. Frauenarzt 1984; 4).

On the other hand gastro-intestinal infections remain a common problemin the human population. Bacterial adherence to the gastrointestinalepithelium has been recognized as an important mechanism in theinitiation and pathogenesis of gastrointestinal tract infections (GH).Many gastrointestinal pathogens which colonize the intestinal tract may,depending on host factors and bacterial virulence factors, expressvirulence characteristics that enable them to resist the normallyefficient host defence mechanisms.

The use of bacteria originating from the autochthonous microflora, likee.g. lactobacilli, to exclude pathogens from colonizing thegastrointestinal tract is a concept which has been studied ratherextensively (see e.g. Alain Servin in “Antagonist activities oflactobacilli and bifidobacteria against microbial pathogens—FEMSMicrobiology Reviews 2004—in press, available on line from sciencedirectwebsite). Some of the lactic acid bacteria strains mentioned in theabove literature have been highlighted for their effect in thegastrointestinal tract and been proposed as possible active agentssuitable for treating various troubles or disorders caused by pathogens,e.g. diarrhoea.

The main goal of a therapy with bacterial agents should be to preventovergrowth of pathogens until such a time that the normal vaginal orintestinal microflora can be re-established. In addition, probiotictherapy is considered as “natural” and without side effects in contrastwith conventional chemical or antibiotic treatments.

Within that context it has been surprisingly observed that lactic acidbacteria strains representative of the healthy human vaginal flora whichexhibited efficiency in the treatment of urogenital infections (seeInternational Patent Application PCT/EP2004/011980 filed on Nov. 22,2004 by Medinova A G, C H-Zurich) were also performing and consequentlyuseful in the prophylactic or therapeutic treatment of intestinalinfections or disorders initiated by gastrointestinal pathogens.

Nowadays, despite of the progresses which have already been madeconcerning the intimate knowledge of lactic acid bacteria (LAB) strains,their properties and their potential use in the probiotic area, therestill remains a need to propose more convenient and more efficientbacteria, namely probiotic bacteria strains to the medical community.

The purpose of this invention is to provide new and useful probioticsparticularly efficient in the treatment of disorders caused by variouspathogens, namely infections or inflammatory diseases of thegastrointestinal tract in mammals, especially humans, respectively ofthe urogenital tract in females, or in the restoration of a balanced andhealthy urogenital or intestinal flora after e.g. severe medicaltreatments like those performed with antibiotics or chemotherapeutics.

The purpose of this invention is to provide as well new methods ofprophylactic or therapeutic treatment of such infections or inflammatorydiseases which involve specifically selected probiotic strains.

SUMMARY OF THE INVENTION

As a first object the invention provides a method for establishing,maintaining or restoring a healthy urogenital flora and environment infemales or a healthy gastrointestinal flora or environment in humans,which comprises administering thereto an effective amount of at leastone probiotic strain of the genus L. acidophilus, L. crispatus, L.gasseri, L. helveticus and L. jensenii selected for their ability tokill urogenital and/or gastrointestinal pathogens and their ability toinhibit internalization of urogenital and/or gastrointestinal pathogenswithin gastrointestinal epithelial cells in combination with a suitabledelivery system.

The invention further provides a method for establishing, maintaining orrestoring a healthy urogenital flora or environment in females prior to,during and/or after pregnancy, which comprises administering thereto aneffective amount of at least one of the above probiotic bacteria strainsin combination with a suitable delivery system.

The invention still further provides a method for preventing or treatingurogenital infections in females or gastrointestinal dysbioses and/orinfections in humans which comprises administering thereto an effectiveamount of at least one of the above probiotic bacteria strains incombination with a suitable delivery system.

The invention still further provides a method for preventing orinhibiting the colonization and/or growth of pathogens in the urogenitaltract of females or in the gastrointestinal tract of humans whichcomprises administering thereto an effective amount of at least oneprobiotic bacteria strains in combination with a suitable deliverysystem.

The invention still further provides a method for modulating a cellularand/or humoral immune response in humans at the vaginal and/orgastrointestinal level, or for inhibiting the inflammatory syndrome, theinfectious syndrome as well as neoplasic processes in humans, whichcomprises administering thereto an effective amount of at least one ofthe above probiotic bacteria strains in combination with a suitabledelivery system.

The invention also relates to probiotic compositions useful in the abovemethods of treatment, more specifically probiotic compositions availablein the form of a food product or a beverage, e.g. those designed forclinical nutrition, a food or beverage composition, a food or beveragesupplement or adjuvant designed either for human or animal consumptionas

Additional and more specific objects of this invention shall appearwithin the course of the description here below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows: A, Table IV—Wallace score; B, Table V—Ameho score.

DETAILED DESCRIPTION OF THE INVENTION

The probiotic bacteria strains used within the frame of this inventionhave been first selected for their ability to adhere to epithelial cellssuch cervix HeLa or Caco-2 which were chosen as models. Cell adhesion isindeed a prerequisite selection feature as it conditions the capacity ofthe said strains to colonize epithelial tissues, e.g. that of theurogenital tract, and then to compete with, inhibit or exclude pathogensadhesion from that specific location.

These strains further exhibit a significant resistance, i.e. survivalrate when exposed to the highly acidic gastric environment and a fairlygood to excellent resistance when exposed to enzymes like pepsin andpancreatin as well; these properties guarantee the necessary survival ofthe selected strains throughout their progression within thegastrointestinal tract.

The said probiotics have been further selected for their additionalability to inhibit adhesion, growth and even survival of pathogens,namely urogenital and gastrointestinal pathogens from epithelial cells.Gram-negative or Gram-positive pathogens such as those mentioned hereafter are representative of those which are significantly affected bythe probiotics of this invention in terms on adhesion, growth orpathogenic activity: Salmonella species, like S. enterica serovarTypmmurium, E. coli, Streptococcus species, e.g. S. agalactiae,Staphylococcus species like S. aureus, Gardnerella species, e.g. G.vaginalis, Prevotella species, e.g. P. bivia; this enumeration is ofcourse not exhaustive.

The probiotic bacteria strains used according to the present inventionhave also the ability to inhibit internalization of pathogens, namelyurogenital or gastrointestinal Gram-negative or Gram-positive pathogenswithin epithelial cells. The most efficient LAB within that frame arethose which express high amounts of both hydrogen peroxide (H₂O₂) andlactic acid in situ where both factors act synergistically. The latterhave indeed proved highly efficient against anaerobic urogenitalpathogens like e.g. Gardnerella and Prevotella species.

The above probiotic bacteria strains, eventually, exhibit a furtherimportant feature, i.e. the ability to modulate the immune response ofimmuno-competent cells, e.g. gastrointestinal mucous membrane cells, inother words the ability to initiate, stimulate or reinforce the immuneresponse of said cells when infected by either gram-negative orgram-positive pathogens like those mentioned here above, e.g. urogenitalpathogenic E. coli. Due to their specific features the said LAB strainshave consequently the capacity to inhibit the inflammatory syndrome ofimmuno-competent cells when exposed to pathogen contamination.

Quite interestingly that specific feature performs the modulation of theimmune response referred to here above using two distinct routes, i.e.via the induction of either pro- or anti-inflammatory cytokines like IL10, respectively, IL 12, TNF or IFN. It has been further observed thatsome LAB strains of this invention exhibit a high IFNγ inductionpotential, namely L. acidophilus KS 116.1 and L. gasseri KS 124.3, afeature which favors the use of same as anti-infectious agents.

That strain specificity provides consequently to the man skilled in theart the possibility to select the most appropriate strain or combinationof strains for performing the medical treatment which is envisaged.

Among the LAB strains which exhibit these properties preferred speciesaccording to this invention are listed here after: L. jensenii KS 109,L. gasseri KS 114.1, L. crispatus KS 116.1, L. jensenii KS 119.1, L.crispatus KS 119.4, L. gasseri KS 120.1, L. jensenii KS 121.1, L.jensenii KS 122.1, L. gasseri KS 123.1, L. gasseri 124.3, L. gasseri KS126.2, L. crispatus 127.1, L. jensenii KS 129.1, L. jensenii KS 130.1,L. helveticus KS 300 and L. acidophilus KS 400. Most of these strainsare also representative of the healthy human vaginal micro flora.

As particularly preferred species, on can further cite the followingstrains:

L. gasseri KS 114.1 (CNCM 1-3482): gram positive—catalasenegative—oxidase negative—lactic acid production 10.5 g/l—H₂O₂production 10 mg/l

API 50 CHI test: positive for GAL, GLU, FRU, MNE, NAG, ESC, SAL, CEL,MAL, SAC, TRE and GEN

negative for: KON, GLY, ERY, DARA, LARA, RIB, DXYL, LXYL, ADO, MDX, SBE,RHA, DUL, INO, MAN, SOR, MDM, MDG, AMY, ARB, LAC5 MEL, INU, MLZ, RAF5AMD, GLYG, XLT, TUR, LYX, TAG, DFUC5 LFUC5 DARL, LARL, GNT, 2KG and 5KG

L. crispatus KS 116.1 (CNCM 1-3483): gram positive—catalasenegative—oxidase positive—lactic acid production 9.6 g/l—H₂O₂ production2 mg/l

API 50 CHI test: positive for GAL, FRU, MNE, NAG, ESC, SAL, MAL and SAC

negative for: KON, GLY, ERY, DARA, LARA, RIB, DXYL, LXYL, ADO, MDX, GAL,SBE, RHA, DUL, INO, MAN, SOR, MDM, MDG, AMY, ARB, CEL, LAC, MEL, TRE,INU, MLZ, RAF, AMD, GLYG, XLT, GEN, TUR, LYX, TAG, DFUC, LFUC, DARL,LARL, GNT, 2KG and 5KG

L. jensenii KS 119.1 (CNCM 1-3217): gram positive—catalasenegative—oxidase negative—lactic acid production 7.4 g/l—H₂O₂ production20 mg/l

API 50 CHI test: positive for GLU, FRU, MNE, NAG, AMY, ESC, SAL, CEL,MAL, MEL, SAC, GEN and TAG—variable for: RIB

negative for: KON, GLY, ERY, DARA, LARA, DXYL, LXYL, ADO, MDX, GAL, SBE,RHA, DUL, INO, MAN, SOR, MDM, MDG, ARB, LAC, TRE, INU, MLZ, RAF, AMD,GLYG, XLT, TUR, LYX, DFUC, LFUC, DARL, LARL, GNT, 2KG and 5KG

L. crispatus KS 119.4 (CNCM 1-3484): gram positive—catalasenegative—oxidase positive—lactic acid production 10.3 g/l—H₂O₂production negative

API 50 CHI test: positive for GAL, GLU, FRU, MNE, NAG, ESC, MAL, LAC,SAC and AMD negative for: KON, GLY, ERY, DARA, LARA, RIB, DXYL, LXYL,ADO, MDX, SBE, RHA, DUL, INO, MAN, SOR, MDM, MDG, AMY, ARB, SAL, CEL,MEL, TRE, INU, MLZ, RAF, GLYG, XLT, GEN, TUR, LYX, TAG, DFUC, LFUC,DARL, LARL, GNT, 2KG and 5KG

L. gasseri KS 120.1 (CNCM 1-3218): gram positive—catalasenegative—oxidase negative—lactic acid production 10.6 g/l—H₂O₂production 1 mg/l

API 50 CHI test: positive for: GAL, GLU, FRU, MNE, AMY, ESC, SAL, CEL,MAL, LAC, SAC, TRE and AMD

negative for: KON, GLY, ERY, DARA, LARA, RIB, DXYL, LXYL, ADO, MDX, SBE,RHA, DUL, INO, MAN, SOR, MDM, MDG, NAG, ARB, MEL, INU, MLZ, RAF, GLYG,XLT, GEN, TUR, LYX, TAG, DFUC, LFUC, DARL, LARL, GNT, 2KG and 5KG

L. jensenii KS 121.1 (CNCM 1-3219): gram positive—catalasenegative—oxidase negative—lactic acid production 10.6 g/l—H₂O₂production 1 mg/l

API 50 CHI test: positive for: GAL, GLU, FRU, MNE, AMY, ARB, ESC, SAL,CEL, MAL, SAC and AMD—variable for: RIB, NAG, LAC, RAF and LFUC

negative for: KON, GLY, ERY, DARA, LARA, DXYL, LXYL, ADO, MDX, SBE, RHA,DUL, INO, MAN, SOR, MDM, MDG, MEL, TRE, INU, MLZ, GLYG, XLT, GEN, TUR,LYX, TAG, DFUC, DARL, LARL, GNT, 2KG and 5KG

L. gasseri KS 123.1 (CNCM 1-3485): gram positive—catalasenegative—oxidase negative—lactic acid production 8.5 g/l—H₂O₂ production10 mg/l

API 50 CHI test: positive for: GLU, MNE, NAG, ESC, MAL and SAC —variablefor RJB and 5KG

negative for: KON, GLY, ERY, DARA, LARA, DXYL, LXYL, ADO, MDX, GAL, FRU,SBE, RHA, DUL, INO, MAN, SOR, MDM, MDG, AMY, ARB, SAL5CEL, LAC, MEL,TRE, INU, MLZ, RAF, AMD, GLYG, XLT, GEN, TUR, LYX, TAG, DFUC, LFUC,DARL, LARL, GNT and 2KG

L. gasseri KS 124.3 (CNCM 1-3220): gram positive—catalasenegative—oxidase negative—lactic acid production 17.0 g/l—H₂O₂production 20 mg/l

API 50 CHI test: positive for: GAL, GLU, FRU, MNE, NAG, ESC, SAL, MAL,SAC and TRE—variable for: RIB, AMD, GEN and 5KG negative for: KON, GLY,ERY, DARA, LARA, DXYL, LXYL, ADO, MDX, SBE, RHA, DUL, INO, MAN, SOR,MDM, MDG, AMY, ARB, CEL, LAC, MEL, INU, MLZ, RAF, GLYG, XLT, TUR, LYX,TAG, DFUC, LFUC, DARL, LARL, GNT and 2KG

L. crispatus KS 127.1 (CNCM 1-3486): gram positive—catalasenegative—oxidase positive—lactic acid production 16.7 g/l—H₂O₂production negative

API 50 CHI test: positive for RIB, GAL, GLU, FRU, MNE, MAN, SOR, NAG,AMY, ESC, SAL, CEL, MAL LAC, SAC, TRE, MLZ, AMD, GLYG, GEN, TAG andGNT—variable for GLY and DXYL

negative for: KON, ERY, DARA, LARA, LXYL, ADO, MDX, SBE, RHA, DUL, INO,MDM, MDG, ARB, MEL, INU, MLZ, RAF, XLT, TUR, LYX, DFUC, LFUC, DARL,LARL, 2KG and 5KG

L. helveticus KS 300 (CNCM 1-3360): gram positive—lactic acid production10.45 g/kg—H₂O₂ production 1 mg/l

API 50 CHI test—positive for: GAL, GLU, FRU, MNE, AMY, ARB, ESC, SAL,CEL, MAL, LAC, SAC, TRE and AMD

negative for: RIB, MAN, GLY, SOR, KON, ERY, MLZ, DARA, LARA, LXYL, ADO,MDX, SBE, RHA, DUL, INO, MDM, MDG, MEL, INU, RAF, TAG, GNT, XLT, TUR,LYX, DFUC, LFUC, DARL, LARL, 2KG and 5KG

These strains have been duly registered at the Pasteur Institute, Paris(France) in accordance with the Budapest Treaty.

According to the present invention, and due to their specificantipathogen activity, the probiotic bacteria strains mentioned hereabove can be used advantageously for preventing or treating urogenitalinfections in females and/or gastrointestinal infections in humans, morespecifically humans, and for preventing or inhibiting the colonizationand/or growth of pathogens in the urogenital tract or environment offemales and in the gastrointestinal tract and environment of humans aswell.

Also, the said probiotic bacteria strains can be used in a quiteefficient way for maintaining or restoring a healthy urogenital and/orgastrointestinal flora in humans, especially humans, in particular aftersevere medical treatments like those performed with antibiotics.

The corresponding therapeutic or prophylactic treatments are performedby administering an effective amount of the selected strain or strainsof this invention in combination with a suitable, if ever necessary foodgrade, delivery system, support or carrier which has been designedtherefor. The terms “therapeutic treatment” used within that contextmostly refer to a combined treatment where the patients have been firstsubject to the administration of relatively “aggressive” chemicals orantibiotics and when the convenient probiotics administration thenoccurs once pathogen eradication has been completed and administrationof antibiotics has been stopped. The term “suitable” is meant to definea delivery system which keeps intact all the properties of the probioticbacteria strains which are used for performing the above treatments

Probiotic compositions according to this invention can further compriseusual LAB growth factors or prebiotics, e.g. dedicated natural growthfactors like skim milk powder (MSK). Said compositions are preferably inthe form of a food product or a beverage, a food or beverage compositionlike e.g. those designed for clinical nutrition, a food or beveragesupplement or adjuvant designed either for human or animal consumption.

Dairy food products or beverages like fermented milks, fresh cheeses oryogurts or their dried or freeze dried equivalents represent suitabledelivery systems. As e.g. food supplement or adjuvant powdered milk ormilk derivatives matrixes loaded with the selected probiotics provedquite convenient. If ever necessary, said powdered matrixes can befurther packaged as e.g. gelatin or cellulose capsules, gelules ortablets.

Said probiotic compositions can further comprise one or several lacticacid, i.e. probiotic or not, bacteria strains of the prior art as welland further additives like pH stabilizers, viscosity stabilizers,preservatives, antioxidants, colorants or flavors.

The compositions referred to here above may contain the selectedprobiotic microorganisms in amounts which can range from about 10⁶ cfu(colony forming units) to about 10¹¹ cfu per g or dose or unit,preferably in a form that keeps their viability and their specificityintact, e.g. in a encapsulated or lyophilized form. The ultimate detailsof said compositions as well as their dosage shall depend, eventually,on the specific application they are intend for, the age or healthstatus of the patients or person the be treated, the nature of thepathogen contamination or the benefit expected from preventiveadministration of the probiotics. It is within current skills andexpertise of the medical or nutritional community to adjust all therelevant parameters.

When compared to prior known reference strains (see examples below) theprobiotic bacteria strains selected within the frame of the presentinvention have shown either a similar or even higher antipathogenactivity depending on the experimental model which has been selectedtherefor.

The following examples illustrate only some of the embodiments of thisinvention and so are not intended to constitute any limitation orrestriction thereof.

EXAMPLES Material and Methods

Tested Srain Code hensenii 109 KS 109 crispatus 116.1 KS 116.1 jensenii119.1 KS 119.1 gasseri 120.1 KS 120.1 jensenii 121.1, KS 121.1 jensenii122.1 KS 122.1 gasseri 123.3 KS 124.3 gasseri 126.2 KS 126.2 jensenii129.1 KS 129.1 L. jensenii 130.1 KS 130.1 helveticus 300 KS 300acidophilus 400 KS 400

The control adhering lactobacilli strain are the L. casei rhamnosusstrain GG (ATCC Accession no 53103), the L. rhamnosus strain GR-I (ATCCAccession no 55826) and the L. fermentum strain RC-14 (ATCC Accession no55845).

AU the lactobacilli strains were grown in De Man, Rogosa, Sharpe (MRS)broth (Biokar Diagnostic, Beauvais, France) for 18 h at 37° C.

Bacterial pathogens. Salmonella enterica serovar Typhimurium strain SL1344 was a gift of B.A.D. Stacker (Stanford, Calif.). Bacteria weregrown overnight for 18 h at 37° C. in Luria broth (Difco Laboratories).

Uropathogenic diffusely-adhering Escherichia coli strains IH1 1128 and7372, and diarrheagenic strain C 1845 were gifts from B. Nowicki (TexasUniversity, Galvestone) and S. Moseley (Seattle University). Strain 7372carriers the class II papG allele, the hly gene (haemolysin) and the Droperon. Strain IH1 1128 carriers the Dr operon. Strain C1845 carriersthe Daa operon. All bacterial strains were maintained on LB plates andprior to infection, bacteria were grown in LB broth at 37° C. for 18 h.

Staphylococcus aureus strain was from the Pasteur culture collection(Paris). Bacteria were grown overnight at 370C in TSA broth (DifcoLaboratories).

Strains of Streptococcus agalactiae DSM 2134, Gardnerella vaginalis DSM4944, Prevotella bivia C1-I (DSM 20514) and Candida albicans DSM 1386were from Medinova Ltd, Zurich, Switzerland). Staphylococcus aureusstrain was from the Pasteur culture collection (Paris). Bacteria weregrown overnight at 37° C. in TSA broth (Difco Laboratories).Streptococcus agalactiae strain was grown overnight at 370C in BHI broth(Difco Laboratories).

Candida albicans strain was grown overnight at 37° C. in Sabouraudbroth. Gardnerella vaginalis was grown on Gardnerella agar platespurchased from BioMerieux France.

Bacteria were suspended in buffered sodium chloride-peptone solution pH7.0 to about 10⁶ colony forming unit (CFU/ml). 500 μl or the preparedsuspensions were spread out on the agar plate. The inoculated plateswere dried under sterile laminar air flow conditions. The agar plateswere then incubated under anaerobic conditions using a sealed anaerobicjar (Becton Dickinson, USA) at 370C for 36° h in maximum. Before use,the Gardnerella vaginalis strain was sub-cultured in BHI supplementedwith yeast extract, maltose and horse serum, under anaerobic conditionsusing a sealed anaerobic jar at 37° C. for 36 h in maximum.

Before use, bacterial cultures were centrifuged at 5.500×g for 5 min at4° C. The culture medium was discarded and the bacteria were washed oncewith phosphate-buffered saline (PBS) and re suspended in PBS.

Cell lines and cultures. Human cervical HeLa cells were cultured at 37°C. in a 5% CO₂-95% air atmosphere in RPMI 1640 with L-glutamine (LifeTechnologies) supplemented with 10% heat-inactivated (30 min, 56° C.)foetal calf serum (FCS; Boehringer, Mannheim, Germany). Cells were usedfor infection assays before confluence, i.e., after 5 days in culture.

The human intestinal cell line used was the TC7 clone (Caco-2/TC7),established from the parental Caco-2 cell line. Cells were routinelygrown in Dulbecco modified Eagle's minimal essential medium (DMEM) (25mM glucose) (Invitrogen, Cergy, France), supplemented with 15%heat-inactivated (30 min, 56° C.) foetal calf serum (Invitrogene) and 1%non-essential amino acids (Invitrogene) as previously described. Formaintenance purposes, cells were passaged weekly using 0.02% trypsin inCa²⁺Mg²⁺-free PBS containing 3 mM EDTA. Experiments and maintenance ofthe cells were carried out at 37° C. in a 10% CO₂/90% air atmosphere.The culture medium was changed daily. Cells were used at post-confluenceafter 15 days of culture (fully differentiated cells) for infectionassay of S. enterica serovar Typhimurium.

Adhesion assays. The adhesion of lactobacilli strains onto cervix HeLacells and intestinal Caco-2/TC7 cells was examined according to thefollowing steps: the cells monolayers were washed twice withphosphate-buffered saline (PBS). For each adhesion assay, 0.5 ml of theLactobacillus suspension (bacteria with spent broth culture supernatant)was mixed with DMEM (0.5 ml), and then added to each well of the tissueculture plate (24 wells) which was then incubated at 37° C. in 10%CO₂/90% air. The final concentrations of bacteria examined were 1×10⁸,2×10⁸, 1×10⁹, and 2×10⁹ bacteria per ml. After 1 h incubation, themonolayers were washed five times with sterile PBS, fixed with methanol,stained with Gram stain, and then examined microscopically under oilimmersion. Each adhesion assay was conducted in duplicate with cellsfrom three successive passages. For each assay, the number of adherentbacteria was determined in 20 random microscopic areas (adhesion score:0 to 5). Moreover, the level of viable adhering lactobacilli wasdetermined by quantitative determination of bacteria associated with theinfected cell monolayers. After being infected, cells were washed twicewith sterile PBS and lysed with sterilized H₂O. Appropriate dilutionswere plated on tryptic soy agar (TSA) to determine the number of viablecell-associated bacteria by bacterial colony counts.

Each cell-association assay was conducted at least in triplicate, withthree successive cell passages. Results were expressed as CFU/ml ofcell-associated bacteria.

Activity against the growth of pathogens. A culture medium containingMRS (5 ml) and specific pathogen culture medium (5 ml) was inoculatedwith 0.1 ml of a cultivated pathogen and 0.1 ml of culturedLactobacillus strain. Control was a culture medium inoculated with 0.1ml of a cultivated pathogen and 0.1 ml of non-cultivated MRS adjusted topH 4.5. At indicated time-points, aliquots were removed, seriallydiluted and plated on tryptic soy agar to determine bacterial colonycounts of pathogen. Each assay was conducted at least in triplicate.Results were expressed as CFU/ml.

Activity against the viability of pathogens. Colony count assays wereperformed by incubating 10⁸ CFU/ml pathogen (0.5 ml) with thelactobacilli culture (10⁸ CFU/ml, 0.5 ml) at 37° C. Control wasnon-cultivated MRS adjusted to pH 4.5. Initially and at predeterminedintervals, aliquots were removed, serially diluted and plated on trypticsoy agar to determine bacterial colony counts of pathogen. Each assaywas conducted at least in triplicate. Results were expressed as CFU/ml.

Inhibition of uropathogenic E. coli adhesion onto epithelial HeLa cells.For cell monolayer infection, pathogens were cultured at 37° C. for 18 hin appropriate culture media as described above. Prior to infection, thecell monolayers, prepared in twenty four-well TPP tissue culture plates(ATGC, Paris, France), were washed twice with PBS. Infecting bacteriawere suspended in the culture medium and a total of 0.5 ml DMEM+0.25 mlculture pathogen (1×10⁸ CFU/ml)+0.25 ml lactobacilli culture (1.5×10⁹CFU/ml) were added to each well of the tissue culture plate. The plateswere incubated at 37° C. in 10% CO₂/90% air for different time ofinfection as indicated and then were washed three times with sterile PBSand lysed with sterilised H₂O. Appropriate dilutions were plated ontryptic soy agar to determine the number of viable cell-associatedbacteria by bacterial colony counts. Each assay was conducted intriplicate with three successive passages of HeLa cells.

Analysis. Results are expressed as means±standard error to the mean. Forstatistical comparisons, Student's t test was performed.

RESULTS Example 1 1. Adhesion Capacity of L. Jensenii KS 119.1 and KS130.1, L. Crispatus KS 116.1 and L. Gasseri KS 124.3 onto HeLa andCaco-2/TC7 Cells

The level of adhesion of the above strains was determined after thecells were inoculated with four concentrations of lactobacilli (5×10⁷;1×10⁸; 5×10⁸; 1×10⁹ CFU/well). Generally, a concentration-dependentadhesion was observed.

In cervix HeLa cells, adhesion levels observed show that all the testedstrains are adhering. The L. jensenii KS 119.1 and KS 130.1 strainsappeared the best adhering strains (7.5 log CFU/ml at 5×10⁸ CFU/well) ascompared with the control adhering strains, L. casei rhamnosus GG and L.rhamnosus GR1 strains.

In intestinal Caco-2/TC7 cells, adhesion levels observed show that allthe Medinova strains are adhering. The L. crispatus KS 116A, L. jensenii119.1, 129.1 and KS 130.1, L. gasseri 124.3 strains appeared the bestadhering strains (7.5-8 logs CFU/ml at 5×10⁸ CFU/well) as compared withthe control adhering strains, L. casei rhamnosus GG and L. rhamnosus GR1strains.

As observed by scanning electron microscopy, all the “inventionlactobacilli strains” appeared adhering in close contact with the HeLaand Caco-2/TC7 cells.

On the basis of their adhering properties, the L. crispatus KS 116.1 andL. jensenii 119.1 have been selected for the following studies ofantibacterial activities against urogenital and intestinal pathogens.

2. Activity of KS 116.1 and KS 119.1 on the Growth of Urogenital andIntestinal Pathogens

It has been examined whether the above mentioned strains are active onthe growth of Staphylococcus aureus, Streptococcus agalactiae,uropathogenic and diarrheagenic E. coli, and diarrheagenic Salmonellaenterica serovar Typhimurium. The growth of pathogens was measured at 5,8, 18 and 24 h.

For Staphylococcus aureus, the control L. rhamnosus strain GR-I and L.fermentum strain RC-14 inhibited the growth of bacteria. Similarly, L.crispatus KS 116.1 and L. jensenii 119.1 inhibited the growth ofStaphylococcus aureus and showed a decrease in the viable bacterianumber. When activities of lactobacilli strains were compared, the L.jensenii 119.1 appeared the most active strain.

For uropathogenic E. coli strains IH1 1128 and 7372, the control L.rhamnosus strain GR-1 and L. fermentum strain RC-14 inhibited the growthof the bacteria. Similarly, L. crispatus KS 116.1 and L. jensenii KS119.1 inhibited the growth of E. coli. When activities of lactobacillistrains were compared, the L. jensenii 119.1 appeared the most activestrain.

For diarrheagenic E. coli strain C1845, the control L. rhamnosus strainGR-I and L. fermentum strain RC-14 inhibited the growth of the bacteria.Similarly, L. crispatus KS 116.1 and L. jensenii KS 119.1 inhibited thegrowth of E. coli. When activities of lactobacilli strains werecompared, the same activity was found for all the lactobacilli strainsexamined.

For diarrheagenic S. enterica serovar Typhimurium strain SL1 344, thecontrol L. rhamnosus strain GR-I and L. fermentum strain RC-14 inhibitedthe growth of the bacteria. Similarly, L. jensenii 119.1 inhibited thegrowth of S. enterica serovar Typhimurium. When activities oflactobacilli strains were compared, the same activity was found for thecontrol L. rhamnosus strain GR-I and L. fermentum strain RC-14 and L.jensenii KS 119.1. In contrast, the L. crispatus KS 116.1 showed a loweractivity.

For Candida albicans no activity was found for the control L. rhamnosusstrain GR-I and L. fermentum strain RC-14, and L. crispatus KS 116.1 andL. jensenii KS 119.1.

3. Killing Activity of KS 116.1 and KS 119.1 Against Urogenital andIntestinal Pathogens

It has been examined whether said lactobacilli are active on theviability of Staphylococcus aureus, Streptococcus agalactiae,uropathogenic and diarrheagenic E. coli, and diarrheagenic Salmonellaenterica serovar Typhimurium. The effect on viability of pathogens wasmeasured at 2, 3, and 4 h.

For Staphylococcus aureus, the control L. rhamnosus strain GR-I and L.fermentum strain RC-14, and L. jensenii 119.1 decreased for 2 logs theviability of bacteria. In contrast, the L. crispatus KS 116.1 showed noactivity.

For Streptococcus agalactiae, the control L. rhamnosus strain GR-I andL. fermentum strain RC-14, and L. jensenii 119.1 and L. crispatus KS116.1 showed no activity.

For uropathogenic E. coli strains IH11128 and 7372, the control L.rhamnosus strain GR-1 and L. fermentum strain RC-14 showed 4 logs ofdecrease in viability of bacteria. L. crispatus KS 116.1 and L. jensenii119.1 were not active showing only one log of decrease in. viability ofthe bacteria.

For diarrheagenic E. coli strain C1845, both of the control L. rhamnosusstrain GR-I and L. fermentum strain RC-14, and L. crispatus KS 116.1 andL. jensenii 119.1 showed a low activity on the viability of C1845bacteria (2 logs of decrease).

For diarrheagenic S. enterica serovar Typhimurium strain SL1344, both ofthe control L. rhamnosus strain GR-I and L. fermentum strain RC-14, andL. crispatus KS 116.1 and L. jensenii 119.1 showed a great activity bydecreasing the viability of SL1344 bacteria (5 logs of decrease).

For Gardnerella vaginalis, the control L. rhamnosus strain GR-I and L.fermentum strain RC-14, and L. fermentum strain RC-14, and L. jensenii119.1 decreased for 2 logs the viability of Gardnerella. In contrast,the L. crispatus KS 116.1 showed no activity.

For Candida albicans no activity was found for the control L. rhamnosusstrain GR-I and L. fermentum strain RC-14, and L. crispatus KS 116.1 andL. jensenii KS 119.1.

4. Inhibition of the Adhesion of Uropathogenic E. Coli Strain IH1 1128Strain onto HeLa Cells by KS 116.1 and KS 119.1.

It has been examined whether said lactobacilli are able to inhibit theadhesion of uropathogenic E. coli strain IH11128 onto HeLa cells. Theeffect of the control L. rhamnosus strain GR-I and L. fermentum strainRC-14, and L. jensenii 119.1 and L. crispatus KS 116.1 was measured atthree concentrations: 1×10⁸, 5×10⁸, and 1×10⁹ bacteria per well.

A 30 to 40% of inhibition of IH11128 adhesion was found at aconcentration of 1×10⁸ bacteria per well for the control L. rhamnosusstrain GR-I and L. fermentum strain RC-14. At this concentration, the L.jensenii KS 119.1 and L. crispatus KS 116.1 were inactive. Inhibition ofIH11128 adhesion was effective at a concentration of 5×10⁸ bacteria perwell for L. jensenii 119.1 and L. crispatus KS 116.1 and a similarinhibition that those obtained with the control L. rhamnosus strain GR-Iand L. fermentum strain RC-14 was observed. A similar high inhibitionlevel of IH11128 adhesion was observed with the control L. rhamnosusstrain GR-I and L. fermentum strain RC-14, and L. jensenii KS 119.1 andL. crispatus KS 116.1 at the concentration of 1×10⁹ bacteria per well.

Example 2 1. Activity of L. Gasseri KS 124.3, L. Helveticus KS 300 andL. Acidophilus KS 400 on the Growth of Urogenital and IntestinalPathogens

It has been examined whether the strains referred to here above areactive against the growth of Staphylococcus aureus, Streptococcusagalactiae, Candida albicans and uropathogenic and diarrheagenic E. colistrains IH11128 and 7372. The growth of pathogens was measured at 5, 8,18 and 24 h.

No activity was developed against Streptococcus agalactiae and Candidaalbicans by L. gasseri KS 124.3, L. helveticus KS 300 and L. acidophilusKS 400 as well as by the control strains GR-I and RC-14. ConcerningStaphylococcus aureus, the control L. rhamnosus strain GR-I and L.fermentum strain RC-14 efficiently inhibited the growth of the bacteria.Similarly, L. gasseri KS 124.3, L. helveticus KS 300 and L. acidophilusKS 400 inhibited the growth of Staphylococcus aureus and showed adecrease in the viable bacteria number. When activities of lactobacillistrains were compared, the L. helveticus KS 300 appeared the most activestrain.

For uropathogenic E. coli strains IH11128, the control strains L.rhamnosus GR-I and L. fermentum RC-14 efficiently inhibited the growthof the bacteria. Similarly, L. helveticus KS 300 efficiently inhibitedthe growth of E. coli. When activities of lactobacilli strains werecompared, a lower activity appeared for L. gasseri KS 124.3 and L.acidophilus KS 400.

For uropathogenic E. coli strain 7372, both control strains L. rhamnosusGR-I and L. fermentum RC-14 strains inhibited the growth of bacteria.Similarly L. helveticus KS 300 inhibited the growth of said bacteriawhereas L. acidophilus KS 400, however, was active only at 25 hours.

2. Killing Activity of KS 124.3, KS 300 and KS 400 Against Urogenitaland Intestinal Pathogens

It has been examined whether said lactobacilli are active on theviability of Staphylococcus aureus, Strteptococcus agalactiae, Candidaalbicans, uropathogenic E. coli IH1 1128 and 7372, diarrheagenic E. coliC1 845 and Gardnerella vaginalis. The effect on viability of pathogenswas measured at 2, 3, and 4 h.

For Staphylococcus aureus, the control strains L. rhamnosus GR-I and L.fermentum RC-14, and L. gasseri KS 124.3, L. helveticus KS 300 and L.acdophilus KS 400 decreased for 2-3 logs the viability of bacteria.

Concerning Streptococcus agalactiae and Candida albicans the two controlstrains and L. gasseri KS 124.3, L. helveticus KS 300 and L. acdophilusKS 400 showed no activity.

For uropathogenic E. coli strains IH11128, the control strains L.rhamnosus strain GR-I and L. fermentum RC-14 and L. helveticus KS 300 aswell showed 3 logs of decrease in viability of the bacteria. L.acidophilus KS 400 and L. gasseri KS 124.3 were not active.

Concerning uropathogenic E. coli strains 7372, the control strainsshowed 2 logs of decrease in viability of the bacteria. L. helveticus KS300 showed 3 logs of decrease whereas L. acidophilus KS 400 and L.gasseri KS 124.3 were not active within the same conditions.

For Gardnerella vaginalis, both control strains, L. acidophilus KS 400and L. gasseri KS 124.3 showed 3 logs of decrease in viability of thebacteria. A rapid and efficient activity was observed for L. helveticusKS 300, higher than that found for the above control strains.

For diarrheagenic E. coli strain C1845, both of the control strains L.rhamnosus GR-I and L. fermentum RC-14 killed the bacteria showing a 3log decrease in the viability of same. Similar effect was observed forL. gasseri KS 124.3 whereas no activity was detected concerning L.acidophilus KS 400. L. helveticus KS 300 exhibits a killing which isdefinitely higher that that observed for the above control strains.

Example 3 1. Killing Activity of L. Jensenii KS 121.1 and KS 122.1, L.Gasseri KS 120.1 and L. Helveticus KS 300 Against Urogenital andIntestinal Pathogens

It has been examined whether said lactobacillus strains are active onthe viability of Streptococcus agalactiae, Candida albicans,uropathogenic E. coli IH11128, Gardnerella vaginalis, Prevotella biviaand Salmonella enterica Typhimurium. The effect on viability ofpathogens was measured at 4 h of contact.

Concerning both Streptococcus agalactiae and Candida albicans none ofthe tested lactobacilli showed an activity.

For uropathogenic E. coli strains IH11128, L. jensenii KS 121.1 and KS122.1 showed no activity whereas, in contrast, L. gasseri KS 120.1decreased efficiently (4 logs) the viability of E. coli in unshakenconditions. L. helveticus KS 300 and the L. fermentum RC-I controlstrain decreased of 2 logs the viability of E. coli in unshakenconditions only.

Concerning Gardnerella vaginalis, both L. jensenii KS 121.1 and KS 122.1showed no activity. In contrast L. gasseri KS 120.1 decreasedefficiently (6 logs) the viability of Gardnerella vaginalis in unshakenconditions; L. helveticus KS 300 showed similar efficiency (4 logs ofdecrease) in unshaken conditions also, whereas control strain showed a 3log of decrease only.

For Prevotella bivia, L. gasseri KS 120.1, L. jensenii 122.1, L.helveticus KS 300 and the control strain L. fermentum RC-14 decreasedviability of the bacteria for 2 logs, in unshaken conditions. L.jensenii KS 121.1 which was highly active against Prevotella bivia inunshaken conditions had lost its activity when tested in shakenconditions.

Concerning Salmonella Typhimurium, L. gasseri KS 120.1 (3 logs), L.jensenii KS 121.1 and KS 122.1, L. helveticus KS 300 and the controlstrain L. fermentum RC-14 were quite active (6 logs of decrease) inunshaken conditions. L. gasseri KS 120.1 remained active even in shakenconditions.

2. Activity of KS 120, KS 121.1, KS 122.1 and KS 300 on the Growth ofGardnerella Vaginalis and Prevotella Bivia

The tests have been performed in both shaken and unshaken conditions.

In unshaken conditions L. jensenii KS 121.1 and KS 122.1 inhibited thegrowth of Gardnerella vaginalis whereas L. gasseri 120.1, L. helveticusand the control strain L. fermentum RC-14 inhibited said activity atstill a higher level.

In shaken conditions L. jensenii KS 121.1 and KS 122.1 and L. helveticusas well have lost their activity, whereas L. gasseri 120.1 remainsactive (2 logs of decrease) against Gardneralla vaginalis.

In both shaken and unshaken conditions L. gasseri KS 120.1, L. jenseniiKS 121.1 and KS 122.1, L. helveticus KS 300 and the control straininhibited the growth of Prevotella bivia at a high level.

3. Inhibition of Adhesion of Gardnerella vaginalis and Prevotella Biviaonto HeLa Cells KS 120.1, KS 121.1 and KS 300

The effect of L. gasseri KS 120.1, L. helveticus KS 300 of the controlstrains L. fermentum RC-14 as well as L. casei rhamnosus GG was measuredat the concentration of 1×10⁹ bacteria per well.

The control L. fermentum RC-14 strain and L. jensenii KS 121.1 decreasedfor 2 logs the level of adhesion of Gardnerella vaginalis on the testedcells. L. gasseri KS 120.1 and L. helveticus KS 300 decreased saidadhesion for 4 logs.

L. jensenii KS 121.1 decreased adhesion of Prevotella bivia for 1 logonly, whereas L. gasseri KS 120.1, L. helveticus KS 300 as well as thecontrol strain RC-14 decreased said adhesion for 2 logs.

4. Inhibition of Adhesion and Internalization of Uropathogenic E. ColiStrain IH1 1128 Strain onto HeLa Cells by KS 120.1, KS 121.1 and KS 300

A strategy often used by extra-intestinal pathogens like E. coli toevade host defence mechanism is to establish a local reservoir withinepithelial cells (M. A. Muvlea in Eschrichia coli. Cell Microbiol. 4,257-271-2002) and cell entry by IH11128 strain appears to be aneffective mechanism for promoting prolonged persistence these pathogensin the urinary tract.

The effect of L. gasseri KS 120.1, L. helveticus KS 300 and of thecontrol strains RC-14 and GG strain was examined concerning the aboveuropathogenic E. coli.: L. jensenii 121.1 decreased for 2 logs the levelof viable internalized E. coli, whereas L. gasseri 120.1, L. helveticusand both the control strains have shown a 4 logs of decrease of theinternalized E. coli.

Example 4 Determination of the Resistance to Pepsin % Survival at To+XMin

4.1 The LAB strains selected for that experiment have been grown in 10ml of MRS broth at 37° C. during 24 hours after which the cell culturewas centrifuged for 5 min at 4000 rpm. The pellet thus obtained was thenwashed 3 times in a PBS buffer (pH 7) and subsequently suspended in 1 mlof said PBS buffer.

4.2 200 μl of the above cell suspension was added to a series of 4 testtubes containing each 1 ml of a filtered pepsin solution at pH 2 and 300μl of aqueous NaCl. Immediately after inoculation (T₀) a 10-folddilution series of 100 μl cell suspension from tube no 1 was made usinga Ringer solution and subsequently plated on MR agar for incubation at37° C. during 24 hours. The same procedure was also performed concerningeach of the remaining tubes, at T+20, T+40 and T+60 min respectively.Corresponding bacteria (CFU) counts are reported in Table I here below:

TABLE I Strain 5 min 20 min 40 min 60 min KS 116.1 81 70 0 0 KS 400 7642 5 0 KS 119.1 67 0 0 0 KS 121.1 97 232 187 222 KS 120.1 79 128 161 189KS 124.3 100 16 2 0 KS 300 75 75 45 4

One observes that L. jensenii KS 121.1 and L. crispatus KS 120.1 strainsare particularly resistant to pepsin, even after a prolonged period.

Example 5 Determination of the Resistance to Pancreatin % Survival atT0+X Min

5.1 The LAB strains selected for that experiment have been grown in 10ml of MRS broth at 37° C. during 24 hours after which the cell culturewas centrifuged for 5 min at 4000 rpm. The pellet thus obtained was thenwashed 3 times in a PBS buffer (pH 7) and subsequently suspended in 1 mlof said PBS buffer

5.2 200 μl of the above cell suspension was added to a series of 5 testtubes containing each 1 ml of a 0.1% porcine pancreatin solution at pH 8and 300 μl of aqueous NaCl. Immediately after inoculation (T₀) a 10-folddilution series of 100 μl cell suspension from tube no 1 was made usinga Ringer solution and subsequently plated on MR agar for incubation at37° C. during 24 hours. The same procedure was also performed concerningeach of the remaining tubes, at T+20, T+40, T+60 and T+120 minrespectively. Corresponding bacteria (CFU) counts are reported in TableII here below:

TABLE II Strain 5 min 20 min 40 min 60 min 120 min KS 116.1 152 135 185181 175 KS 400 107 160 127 93 206 KS 119.1 78 113 140 160 151 KS 121.165 48 63 110 55 KS 120.1 181 167 119 129 116 KS 124.3 50 88 119 129 116KS 300 71 80 86 93 71

One observes that L. acidophilus KS 116.1 and KS 400, as well as L.jensenii KS 119.1, L. crispatus 120.1 and KS 124.3 strains, but to alesser extent, are particularly resistant to pancreatin. Quiteinterestingly L. crispatus KS 120.1 is resistant to both pepsin andpancreatin.

Example 6 Modulation of the Immune Response In Vivo Test Using HumanPMBC

The following strains have been tested within the conditions sethereafter concerning their ability to induce or modulate or affect animmune response, more specifically their ability to induce the secretionof cytokines and the like: L. crispatus KS 116.1, L. jensenii 119.1, L.jensenii KS 121.1 and KS 122.1, L. gasseri KS 120.1, L. gasseri KS124.3, L. helveticus KS 300 and L. acidophilus KS 400.

The detection of the induction of cytokines was made by means of a testfor in vitro stimulation of isolated peripheral blood mononuclear cells(PBMC). Among the cytokines induced during these tests, there areinterleukins 10 and 12 (IL10 & IL 12), γ-interferon (γ-IFN) and tumornecrosis factor α (TNFα).

Experimental Procedures

PMBC preparation: Fresh human blood obtained for healthy subjects (fourdonors) was diluted at a 1:2 ratio with PBS-Ca (GIBCO) and purified on aFicoll gradient (GIBCO). After centrifugation at 400×g for 30 min at 20°C. the peripheral blood monocular cellular cells (PMBC's) formed aninterphase ring layer in the serum. PMBC's were aspired carefully,suspended to a final volume of 50 ml using PBD-Ca and washed three timesin the same buffer with centrifugation steps at 350×g for 10 min at 20°C.

PMBC's were subsequently resuspended using complete RPMI medium(GIBCOP), m supplemented with 10% w/v L-glutamine (GIBCO) and gentamycin(150 μg/ml) (GIBCO). PBMC's were counted under the microscope andadjusted at a concentration of 2×10⁶ cells/ml and distributed (in 1 mlaliquots) in 24-well tissues culture plates (Corning, Inc.).

Bacteria preparation: overnight LAB cultures were washed twice with PBSbuffer, pH 7.2 before being resuspended in PBS at concentration of 2×10⁹cfu/ml.

PMBC incubation: from these suspensions 10 μl was transferred into wellsof the PMBC plates which were incubated at 37° C. in a 5% CO₂/95% airatmosphere. After 24 hours incubation the supernatant was aspirated,centrifuged at 2000 rpm and the supernatant removed and stored at −20°C. The control consisted of bacteria-free buffer.

Cytokine quantification: cytokine expression levels have been determinedby ELISA tests (<<Enzyme linked immuno sorbent assay>>). ELISA platesare coated with anti-cytokine antibody (overnight procedure) and theantibody is blocked with PBS/BSA 1%. A proper standard was prepared withknown concentrations of cytokines, covering the dectection range of15,62 to 2000 pg/ml (incubated overnight).

The anti-cytokine detection and quantification was performed with thestreptavidine reaction on substrate (TMB Pharmigen). The commercial kitsPharmigen have been used according to the manufacturer's description.Four cytokines were determined: the pro-inflammatory/Th 1 cytokinesTNFα, IFNγ, IL 12 and the anti-inflammatory/Th 2 cytokine IL10.

TABLE III IL10 IL 12 TNFα IFNγ IL10/IL12 Control 31.25 31.25 31.25 31.251 KS 120.1 1228.67 176.32 17698.83 3513.36 6.96840971 KS 121.1 2297.8747.66 14180.66 897.65 48.2138061 KS 116.1 2856.26 167.6 33569.91 7209.3317.0540573 KS400 3177.49 103.85 26799 6949.13 30.5969186 KS 300 2290.4759.7 18703.66 10047.75 38.3663317 KS 119.1 307.13 198.47 6693.3 9192.741.54748829 KS 124.3 2969.02 660.98 31307.71 16985.56 4.49184544

Observations

a high level of TNFα induction for all the tested LAB strains

a relatively low level of INFγ concerning L. jensenii KS 121.1

the highest IL10 induction potential concerning L. crispatus KS 116.1and KS 400

in contrast to the two L. jensenii strains the two L. gasseri strainshave shown a similar profile, especially when considering the ratio's inIL10/IL12 and in TNFα/INFγ.

Within the above testing frame it is clear that the cytokine inductionprofile is strain specific.

Example 7 Determination of the Anti-Inflammatory Activity (In Vivo TestUsing an Animal Model)

An acute model of mice has been adapted from Camoglio et al. (see Eur.J. Immunol. 2000) where the animals have been fed from day −5 to day +2with selected lactic acid bacteria strains, at a rate of 10⁸ bacteriaper mouse per day. TNBS was then injected on day zero, at a rate of 120mg/kg mice in order to induce acute colitis and the animals have beensacrificed at day +2 and eventually subjected to both macroscopic(Wallace score—Table IV) and histological (Ameho score—Table V) scoring(FIG. 1).

These tables clearly show that the selected lactic acid bacteria strainsexhibit a significant anti-inflammatory effect when compared toreference strains.

Example 8 Probiotic Composition for Local Administration 8.1 VaginalCapsules

Samples of the LAB strains of this invention (see above) have beencultured for min. 24 hours in conditions similar to those mentioned hereabove. The cultured strains have been isolated, washed and lyophilizedindividually, individually suspended in a lactose/MSK powder mixture andeventually divided into unit doses each of them containing about 10⁸-10⁹cfu (colony forming units). Said unit doses have been then poured intogelatin vaginal capsules each of them comprising about 10⁸-10⁹ cfu ofselected LAB strains of this invention.

8.2 Vaginal Suppositories

Soft vaginal suppositories have been prepared using the followingingredients:

buffered lactic acid solution—lactose

PEG 4000

PEG 600

The adequate amount of selected lyophilized LAB strains of thisinvention has been then added to unit doses to afford vaginalsuppositories each comprising about 10⁸-10⁹ cfu.

Example 9 Composition for Oral Administration 9.1 Food Supplement

Edible cellulose capsules (hydroxypropyl methyl cellulose) eachcomprising about 10⁸-10⁹ cfu of selected LAB strains of this inventionhave been manufactured using filler comprising the followingingredients:

dehydrated yoghurt powder

anhydrous dextrose potato starch

microcrystalline cellulose

selected lyophilized LAB strain.

9.2 Fermented Milk Product (Yoghurt)

Portions of a so called “Yoghurt Nature Light” have been prepared usingthe following process: to a batch of standardized 1.5% fat milk therewas added 3% of skimmed milk powder (MSK) and the whole was thenpasteurized at 90° C. for 30 minutes. 1% volume of commercial startercultures of L. bulgaricus and S. thermophilus have been added to thepasteurized milk; then the whole was gently stirred at room temperature,disposed in 100 ml containers which were all eventually incubated at 40°C. during around 4 hours to afford the desired pH.

Then portions of selected lyophilized LAB strains of this invention wereadded to the yoghurt cans in such an amount to have about 10⁸-10⁹ cfuper ml yoghurt can and a further incubation was carried out for about 30min. until to afford a pH of about 4.5 to 4.7. These yoghurt portionscan be stored at 4° C. before consumption.

1. Method for establishing, maintaining or restoring a healthyurogenital flora and environment in females or a healthygastrointestinal flora or environment in humans throughout life, whichcomprises administering thereto a probiotic composition comprising aneffective amount of at least one probiotic strain of the genus L.acidophilus, L. crispatus, L. gasseri, L. helveticus and L. jenseniiselected for their ability to kill urogenital and/or gastrointestinalpathogens and their ability to inhibit internalization of urogenitaland/or gastrointestinal pathogens within urogenital an/orgastrointestinal epithelial cells in combination with a suitabledelivery system.
 2. Method for preventing or inhibiting adhesion,colonization and/or growth of pathogens in the urogenital tract offemales or in the gastrointestinal tract of humans, which comprisesadministering thereto a probiotic composition comprising an effectiveamount of at least one probiotic strain of the genus L. acidophilus, L.crispatus, L. gasseri, L. helveticus and L. jensenii selected for theirability to kill urogenital and/or gastrointestinal pathogens and theirability to inhibit internalization of urogenital and/or gastrointestinalpathogens within urogenital an/or gastrointestinal epithelial cells incombination with a suitable delivery system.
 3. Method for preventing ortreating urogenital infections in females or gastrointestinal dysbiosesand/or infections in humans, which comprises administering thereto aprobiotic composition comprising an effective amount of at least oneprobiotic strain of the genus L. acidophilus, L. crispatus, L. gasseri,L. helveticus and L. jensenii selected for their ability to killurogenital and/or gastrointestinal pathogens and their ability toinhibit internalization of urogenital and/or gastrointestinal pathogenswithin urogenital an/or gastrointestinal epithelial cells in combinationwith a suitable delivery system.
 4. Method for modulating a cellularand/or humoral immune response in humans at the vaginal and/orgastrointestinal level, which comprises administering thereto aprobiotic composition comprising an effective amount of at least oneprobiotic strain of the genus L. acidophilus, L. crispatus, L. gasseri,L. helveticus and L. jensenii selected for their ability to killurogenital and/or gastrointestinal pathogens and their ability toinhibit internalization of urogenital and/or gastrointestinal pathogenswithin urogenital an/or gastrointestinal epithelial cells in combinationwith a suitable delivery system.
 5. Method for inhibiting theinflammatory syndrome, the infectious syndrome as well as neoplasicprocesses in humans, which comprises administering thereto a probioticcomposition comprising an effective amount of at least one probioticstrain of the genus L. acidophilus, L. crispatus, L. gasseri, L.helveticus and L. jensenii selected for their ability to kill urogenitaland/or gastrointestinal pathogens and their ability to inhibitinternalization of urogenital and/or gastrointestinal pathogens withinurogenital an/or gastrointestinal epithelial cells in combination with asuitable delivery system.
 6. The method according to any of claims 1 to5 wherein the lactic acid bacteria strains are selected from the groupconsisting of L. crispatus KS 116.1 (CNCM I-3483), L. crispatus KS 119.4(CNCM I-3484), L. crispatus 127.1 (CNCM I-3486), L. gasseri KS 114.1(CNCM I-3482), L. gassed KS 120.1 (CNCM I-3218), L. gasseri KS 123.1(CNCM I-3485), L. gasseri KS 124.3 (CNCM I-3220), L. helveticus KS 300(CNCM I-3360), L. jensenii KS 119.1 (CNCM I-3217) and L. jensenii KS121.1 (CNCM I-3219).
 7. The method according to any of claims 1 to 6wherein the suitable delivery system further comprises lactic acidbacteria (LAB) growth factors and/or prebiotic ingredients.
 8. Themethod according to any of claims 1 to 7 wherein the suitable deliverysystem is designed for local, namely intra-urethral, vaginal or analadministration.
 9. The method according to any of claims 1 to 7 whereinthe suitable delivery system is designed for oral administration. 10.The method according to claim 9 wherein the suitable delivery system isa food product or a beverage, a food or beverage composition, a food orbeverage supplement or adjuvant dedicated to human consumption.
 11. Aprobiotic composition useful for establishing, maintaining or restoringa healthy urogenital flora and environment in females or a healthygastrointestinal flora or environment in humans throughout life, whichcomprises administering thereto a probiotic composition comprising aneffective amount of at least one probiotic strain of the genus L.acidophilus, L. crispatus, L. gassed, L. helveticus and L. jenseniiselected for their ability to kill urogenital and/or gastrointestinalpathogens and their ability to inhibit internalization of urogenitaland/or gastrointestinal pathogens within urogenital an/orgastrointestinal epithelial cells in combination with a suitabledelivery system.
 12. A probiotic composition useful for preventing ortreating urogenital infections in females or gastrointestinal dysbiosesand/or infections in humans, which comprises administering thereto aprobiotic composition comprising an effective amount of at least oneprobiotic strain of the genus L. acidophilus, L. crispatus, L. gassed,L. helveticus and L. jensenii selected for their ability to killurogenital and/or gastrointestinal pathogens and their ability toinhibit internalization of urogenital and/or gastrointestinal pathogenswithin urogenital an/or gastrointestinal epithelial cells in combinationwith a suitable delivery system.
 13. A probiotic composition useful forpreventing or treating urogenital infections in females orgastrointestinal dysbioses and/or infections in humans, which comprisesadministering thereto a probiotic composition comprising an effectiveamount of at least one probiotic strain of the genus L. acidophilus, L.crispatus, L. gassed, L. helveticus and L. jensenii selected for theirability to kill urogenital and/or gastrointestinal pathogens and theirability to inhibit internalization of urogenital and/or gastrointestinalpathogens within urogenital an/or gastrointestinal epithelial cells incombination with a suitable delivery system.
 14. A probiotic compositionuseful for inhibiting the inflammatory syndrome, the infectious syndromeas well as neoplasic processes in humans, which comprises administeringthereto a probiotic composition comprising an effective amount of atleast one probiotic strain of the genus L. acidophilus, L. crispatus, L.gasseri, L. helveticus and L. jensenii selected for their ability tokill urogenital and/or gastrointestinal pathogens and their ability toinhibit internalization of urogenital and/or gastrointestinal pathogenswithin urogenital an/or gastrointestinal epithelial cells in combinationwith a suitable delivery system
 15. A probiotic composition according toany of claims 11 to 14 wherein the lactic acid bacteria strains areselected from the group consisting of L. crispatus KS 116.1 (CNCMI-3483), L. crispatus KS 119.4 (CNCM I-3484), L. crispatus 127.1 (CNCMI-3486), L. gasseri KS 114.1 (CNCM I-3482), L. gasseri KS 120.1 (CNCMI-3218), L. gasseri KS 123.1 (CNCM I-3485), L. gasseri KS 124.3 (CNCMI-3220), L. helveticus KS 300 (CNCM I-3360), L. jensenii KS 119.1 (CNCMI-3217) and L. jensenii KS 121.1 (CNCM I-3219).
 16. The compositionaccording to any of claims 11 to 15 wherein the suitable delivery systemfurther comprises lactic acid bacteria (LAB) growth factors and/orprebiotic ingredients.
 17. The composition according to any of claims 11to 16 wherein the suitable delivery system is designed for local, namelyintra-urethral, vaginal or anal administration.
 18. The method accordingto any of claims 11 to 16 wherein the suitable delivery system isdesigned for oral administration.
 19. The method according to claim 18wherein the suitable delivery system is a food product or a beverage, afood or beverage composition, a food or beverage supplement or adjuvantdedicated to human consumption.
 20. The use of at least one lacticbacteria strain of the genus L. acidophilus, L. crispatus, L. gasseri,L. helveticus and L. jensenii selected for their ability to killurogenital and/or gastrointestinal pathogens and their ability toinhibit internalization of urogenital and/or gastrointestinal pathogenswithin urogenital and/or gastrointestinal epithelial cells, for thepreparation of a probiotic composition according to any of claims 11 to14.
 21. The use according to claim 20 wherein lactic acid bacteriastrains are selected from the group consisting of L. crispatus KS 116.1(CNCM I-3483), L. crispatus KS 119.4 (CNCM I-3484), L. crispatus 127.1(CNCM I-3486), L. gasseri KS 114.1 (CNCM I-3482), L. gasseri KS 120.1(CNCM I-3218), L. gasseri KS 123.1 (CNCM I-3485), L. gasseri KS 124.3(CNCM I-3220), L. helveticus KS 300 (CNCM I-3360), L. jensenii KS 119.1(CNCM I-3217) and L. jensenii KS 121.1 (CNCM I-3219).